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RF3 Actions 10/27/22

Hello HOPEians,

Please find below the summary and action items from the RF3 meeting on October 27, 2022. If you have any questions about the meeting or action items please reach out to Dr. Melanie Ott and Dr. Priti Kumar. 

Updates:

Priti Kumar shared that her lab has started some spironolactone experiments with mice, in collaboration with the Valente lab about using pellets. In a couple of months, they will look at rebound for the silencing experiments. She has some CURE infection data with ART (only looking at nanoluciferase). 

Micahel Corely discussed that they are running new single-cell studies. They are flash-freezing tissues from mice using liquid nitrogen. He shared that the brain is the hardest to get sequencing from. Nuclei from the tissues can be isolated and it looks like it is working. 

Ulrike Lange started to test the nanobody CD4 and found transfection is successful in T cells.  Martin started experimenting with transfecting cell lines compared to primary cells and found less transfection in the primary cell system. They have been producing new batches of the nanobodies and the first batch of purified CD4-Nanobody AB6 is to be made and sent to Priti.  Priti mentioned that promoter selection will be key to determining efficacy in Rhesus macaques versus mice.  Ulrike’s group is preparing a summary of cell line models of HIV latency to test Brec1 suppression of HIV.  Ulrike will initiate MTA with Lish’s group for material transfer.  Michael proposed an experiment where GFP+/- JLAT cells were sorted prior to BREC1 transfection to test the recombinase efficacy in active and “latent” cells.  Ulrike’s group is preparing a summary of cell line models of HIV latency to test Brec1 suppression of HIV.  Ulrike will initiate MTA with Lish’s group for material transfer.  Michael proposed an experiment where GFP+/- JLAT cells were sorted prior to BREC1 transfection to test the recombinase efficacy in active and “latent” cells.

Niren Murthy updated on his LNP work.  He mentioned that negatively charged lipids can increase spleen localization.  Although this is known in non-humanized mice this effect is unknown in humanized mice.  In collaboration with the Wang lab at UC-Davis, they put IV injected neg-LNP/CRE mRNA into adult Ai9 mice (n=3).  30 minutes post-injection, spleen cells were analyzed by flow cytometry. Transfection in mice with negatively charged lipids indicates CD3+ T cells are still transfected (7.9% & 8.3% of CD3+ T cells received and expressed the transgene). Lish asked about myeloid cell transfection.  CD11C+ cells did not receive/express the transgene.  Transfection of primary human T cells with aptamer targeting LNPs is being tested for gene delivery to primary human T cells.  Preliminary data show little toxicity. Although the two formulations only showed ~1% transfection) increased doses can be tried.

Melanie Ott and Francisco Belinchon shared information about his transfection experiment and there was not a high enough efficiency with Jurkat cells. Francisco completed Brec1 (BrecOff from Ulrike) transfection in JLATs using nucleofection.  ~0.2% transfection efficiency but no effect on HIV suppression was seen.  Repeats are being done with lentiviral vectors.  Niren mentioned Brec1 is finicky. Permanent expression may be necessary and higher levels of Brec1 are required for efficacy.

-Zoom call between Ulrike and Francisco is needed next week.  

-Western blots to verify Brec1 protein expression are needed.  

-Sorting cells may be one way to delineate the effect of BrecOff in HIV suppression in JLATs.

-Francisco is still optimizing/testing the Piggyback vector and CRISPRoff system, taken over from Martin.

 -Doxycycline inducible vectors to be sent to Francisco from Ulrike’s group (maps and construct).

Actions:

  • Michael – Share mouse tissue flash freezing protocols with Priti (test materials sent)

  • Niren – Send LNPs to Priti for the Kumar lab to test – Transfection of primary human t cells with aptamer target LNPs with heart tissues

  • Ulrike meeting with Francisco to look at data from a previous student for Brec off suppression

    • Ulrike-Docs inducible vectors, prepping DNA, send maps and constructs to Francisco

    • Send new AV to Priti

  • Lish – work on MTA for this (Brec, latency models), Ulrike to send MTA contact officer to Lish

Announcements:

  • If you are attending the Miami Meeting and would like to join the HOPE dinner on 12/12 Please let Robert and me know. 

  • The HOPE Collaboratory-wide meeting is on November 21st.

  • The next RF3 Meeting is December 5th. Please come prepared with some updates. 

  • Please Acknowledge HOPE & the NIH in any publications or presentations and notify the Program Manager. Here are some examples of what you can include. 

“Research reported in this publication was supported by the NIAID of the National Institutes of Health under award number UM1AI164559, with co-funding support from NIDA, NIMH, NHLBI, the NIDDK, and the NINDS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

“This research was supported by NIAID award number UM1AI164559, co-funded by NHLBI, NIDA, NIMH, NINDS, and NIDDK.”

RF3 Actions 6/6/22

Hello HOPEians,

In Monday’s meeting on June 6th, we heard data updates from the Chemnitz and Lange Labs. They explained the construction and functional characterization of an enzymatically dead Brec1 and the generation of different expression plasmids for brec1 and dBrec1. They also went over CD4-specific AAV2 vectors using nanobody capsid engineering. Priti gave a quick update on using CRISPR-BE (BE4) targeted to TAR to inhibit HIV; initial LTR reporter assays show reduced luciferase expression, next they will test using Ya-chi Ho’s clonal cell lines. Priti shared the paper “Efficient viral delivery of Cas9 into human safe harbor” and Nadia shared “Why drug delivery is the key to new medicines”.

There is a working group meeting on CRISPRoff efforts and relevant data on June 13th at 10am PT. Joining information is on the HOPE calendar.

Here are the updated actions from our last meeting. If you have any questions about the meeting or action items please reach out to Melanie and Priti. 

Actions:

  • (In progress) Priti have a meeting with Ulrike 
    • Discuss CD7 nanobodies and adding RSV molecules to AAV for targeted delivery
  • Jan – Generate a Brec1 construct using an SFFV promoter for Priti

Long-term Actions:

  • (In progress) Priti – Provide mouse tissues to Lish for scRNA-seq
    • Lish to set up a pipeline for sequencing and analysis
    • Potentially going to Yale to do initial steps on site
  • Priti- Continue to work on Brec1 delivery using a lenti or VLP based methods

Announcements:

  • The July 4th meeting is canceled for the holiday
  • The next meeting is scheduled for Aug 1st and we will host a HOPE – CRISPR for Cure Joint Meeting with Guest Speakers – Dr. Kamel Khalili and Dr. Tricia Burdo

RF3 Actions 5/3/22

Hello HOPEians,

In yesterday’s meeting, Niren Murthy gave updates about the LNP delivery systems developed in his laboratory with the goal of transfecting mRNAs in human T cells and HSCs. Stephen Yeung gave an update on the surface markers subgroup meeting where it was decided to focus on PD1 and TIGIT as markers of cells with latent HIV.

For our next meeting on June 6th, we will hear data updates from Chemnitz and Lange Labs. Here are updated actions from our meeting in May, we will discuss these at the next meeting. If you have any questions about the meeting or action items please reach out to Melanie and Priti. 

Actions:

  • (In progress) Priti have a meeting with Ulrike 
    • Discuss CD7 nanobodies and adding RSV molecules to AAV for targeted delivery
  • Jan – Generate a BREC1 construct using an SFFV promoter for Priti
  • (In progress) Ulrike – set up a meeting with Warner to discuss good CRISPRi guides
  • All – If you have not already, please complete the HOPE Member Introduction form

Long-term Actions:

  • (In progress) Priti – Provide mouse tissues to Lish for scRNA-seq
    • Lish to set up a pipeline for sequencing and analysis
    • Potentially going to Yale to do initial steps on site
  • Priti- Continue to work on Brec1 delivery using a lenti or VLP based methods

Announcements:

  • The next meeting is scheduled for June 6th. Chemnitz and Lange labs to present.
  • The email hope-rf3-members@gladstone.ucsf.edu can be used to reach the whole RF3 group
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form.

RF3 Actions 4/4/22

​​Hello HOPEians,

In yesterday’s RF3 meeting, we went over past actions items with the following updates:

  1. Phil Norris shared HIV-specific CD4+ granzyme clones with Ndhlovu and Nixon labs; they are following up with the function of these clones.
  2. Chemnitz and Lange labs working on defective BREC; constructs are made and they are testing the functionality of these constructs. They will provide an update on progress at the RF3 meeting on June 6th.
  3. Murthy lab testing lipid nanoparticle delivery in PBMCs, initial tests show low transfection efficiency, they are working on new formulations and testing in mouse models. Currently focusing on mRNA delivery instead of protein delivery of BREC due to difficulties in purification. They will provide an update on progress and discuss CAR-T cell delivery at the next meeting on May 2nd.
  4. Greene lab updated on CRISPRi work. The probability of the suppression is only good while the Cas is physically obstructing the promoter.
  5. The use of CD4 nanobodies for delivery is on pause because the molecule is not endocytosed, Priti will follow up on possibly using a CD7 nanobody. 

For our next meeting on May 2nd, we will resume our normal round table format and hear data updates from Niren. Here are updated actions from our meeting in March, we will discuss these at the next meeting. If you have any questions about the meeting or action items please reach out to Melanie and Priti. 

Actions:

  • Priti set up a meeting with Ulrike
    • Discuss CD7 nanobodies and adding RSV molecules to AAV for targeted delivery
  • Sydney – Set up a single-cell RNA sequencing meeting
    • Invitees: Lish, Melanie, Nadia, Priti
  • Jan – Generate a BREC1 construct using an SFFV promoter for Priti
  • Melanie – send Priti a Tat expression vector using EF1a promoter (WT & defective mutant)
  • Melanie Lab – Find  a good Tat antibody for ChIP
  • Ulrike – set up a meeting with Warner to discuss good CRISPRi guides
  • All – If you have not already, please complete the HOPE Member Introduction form

Long-term Actions:

  • (In progress) Priti – Provide mouse tissues to Lish for scRNA-seq
    • Lish to set up a pipeline for sequencing and analysis
    • Potentially going to Yale to do initial steps on site
  • Priti- Continue to work on Brec1 delivery using a lenti or VLP based methods

Announcements:

  • The next meeting is scheduled for May 2nd.
  • The email hope-rf3-members@gladstone.ucsf.edu can be used to reach the whole RF3 group
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form.

Presenters for next meeting: 

  • 5/2 – Murthy Lab and update on cell surface markers from Stephen (Ndhlovu Lab)
  • 6/6 – Update from Chemnitz and Lange Labs

RF3 Actions 3/7/22

Hello HOPEians,

In today’s RF3 meeting, we had an external speaker, Dr. Ya-Chi Ho from Yale. She gave a presentation titled ‘The clonal expansion dynamics of HIV-1-infected cells revealed by single-cell multiomics’.

For our next meeting on April 4th, we will resume our normal round table format. Here are actions from our meeting in February, we will discuss these at the next meeting. If you have any questions about the meeting or action items please reach out to Melanie and Priti. 

Actions:

  • Doug – Ask Phil Norris for the CD4+ granzyme positive clones
  • (In progress) Jan send Niren contacts and protocol for Brec purification
    • Jan – Send update on defective Brec and send material for ChIP to Melanie
  • Hesong – Work with Bharath and Priti on lipid nanoparticles in primary cells and mice
  • (In progress) Martin Hamann/Ulrike – Send Niren the expression vector for the nanobodies
  • Priti – Find out if it is possible to get access to Ya-chi Ho’s scRNA-seq data
  • Priti or Niren present at the gene therapy amfAR meeting (March 25-27)
  • All – If you have not already, please complete the HOPE Member Introduction form

Long-term Actions:

  • (In progress) Priti – Provide mouse tissues to Lish for scRNA-seq
    • Lish to set up a pipeline for sequencing and analysis
  • Priti- Continue to work on Brec1 delivery using a lenti or VLP based methods

Announcements:

  • The next meeting is scheduled for April 4th.
  • The email hope-rf3-members@gladstone.ucsf.edu can be used to reach the whole RF3 group
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form.

RF3 Actions 2/7/22

Hello HOPEians,

In yesterday’s RF3 meeting, Jan gave an overview of the development and clinical testing of Brec1 and there was an update from Niren on the delivery of mRNA using lipid nanoparticle formations.

Please find below the action items from the RF3 meeting on February 7th.  If you have any questions about the meeting or action items please reach out to Melanie and Priti. 

Actions:

  • Priti or Niren present at the gene therapy amfAR meeting (March 25-27)
  • Jan send Niren contacts and protocol for Brec purification
    • Jan – Send update on defective Brec and send material for ChIP to Melanie
  • Hesong – Work with Bharath and Priti on lipid nanoparticles in primary cells and mice
  • (In Progress) Martin Hamann/Ulrike – Send Niren the expression vector for the nanobodies
  • The surface marker subgroup meeting is scheduled for Feb 10th at 10am PT (1pm ET). Invitees include Lish (Stephen Yueng), Melanie (Bharath), Priti, Nadia, Niren, and Danielle.
  • Priti – Find out if it is possible to get access to Ya-chi Ho’s scRNA-seq data
  • All – If you have not already, please complete the HOPE Member Introduction form

Long-term Actions:

  • (In progress) Priti – Provide mouse tissues to Lish for scRNA-seq
    • Lish to set up a pipeline for sequencing and analysis
  • Priti- Continue to work on Brec1 delivery using a lenti or VLP based methods

Announcements:

  • The next meeting is scheduled for March 7th.
  • The email hope-rf3-members@gladstone.ucsf.edu can be used to reach the whole RF3 group
  • Please keep Sydney and Danielle in the loop when scheduling subgroup meetings
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form.

RF3 Actions 12/6/21

Hello RF3 Terminators,

Please find below the action items from the RF3 meeting on December 6th. Note that the January 3rd RF3 meeting is canceled for the holidays. If you have any questions about the meeting or action items please reach out to Melanie and Priti. 

Actions:

  • Martin Hamann/Ulrike – Send Niren the expression vector for the nanobodies
  • Melanie – Set a subgroup meeting to discuss potential known surface markers for latently infected cells. 
    • Meet in January – Lish, Melanie (Bharath), Priti, Nadia, Niren
  • Priti – Provide mouse tissues to Lish for scRNA-seq
    • Lish to set up pipeline for sequencing and analysis
  • Priti – Find out if it is possible to get access to Ya-chi Ho’s scRNA-seq data
  • Lish and Melanie – Follow up on a computational team for HOPE
    • Melanie will set a meeting with Lish, Melanie, Sydney, and Danielle
  • Niren – Send Doudna paper (in vitro Cas9) to Priti
    • Discuss in vitro binding assays or ChIP assays to confirm if TAR is accessible for base editing guides 
  • Priti – Double check that the protocols are in the dropbox (protocols/antibody list for staining of CD7)
  • Ulrike and Melanie – Check if have LTR-Tat reporter constructs (particularly anything in frame with Gag) and let Priti know for use to test CRISPR guides 
  • All – If you have not already, please complete the HOPE Member Introduction form.

Long-term Actions:

  • Jan – Construct and test if dead Brec1 works
  • Priti- Continue to work on Brec1 delivery using a lenti or VLP based methods

Announcements:

  • The email hope-rf3-members@gladstone.ucsf.edu can be used to reach the whole RF3 group
  • Please keep Sydney and Danielle in the loop when scheduling subgroup meetings
  • Share announcements and achievements for the HOPE Twitter or Website by filling out the Social Media Shoutout form. 

RF3 Actions 11/1/21

Hello HOPEians,

Here are some action items from our RF3 meeting today, Monday, Nov 1, 2021.

Actions Items:

  • Bharath and Daniela – Contact Hesong (Murthy Lab) ASAP about MRN delivery experiments in PBMCs
    • Priti – Send protocol for detecting luciferase by flow
  • Priti – Double check that the protocols are in the dropbox (protocols/antibody list for staining of CD7)
  • Daniela (Ott Lab) – Continue to generate a document on what JLats are available
  • Andrew – Set up second subgroup meeting in November (prep for Dec 6 presentation)
  • Jan – Construct and test if dead Brec1 works
  • Jan – Send paper to Andrew (Brec1 showed 95% specificity for multiple HIV sequences)
  • Ulrike and Melanie – Look for LTR-Tat constructs and anything in frame with Gag
  • Melanie – Talk to Pollard and Korber labs to see if they would like to collaborate
  • Priti- Continue to work on Brec1 delivery using a lenti or VLP based methods

Please make sure to copy Danielle Lyons and Sydney Norman on emails so that we can assist in uploading documents to Dropbox. Emails to the full RF3 group can be made to HOPE-RF3-members@gladstone.ucsf.edu 

The next meeting will be on Dec 6th; Priti will present on humanized mouse models. Andrew will give an update on the subgroup meetings.  

RF3 Actions 10/4/21

Hello HOPEians,

Here are some action items from our last RF3 meeting Monday, October 4, 2021.

Action Items:

  • Danielle/Sydney to set up and share the Dropbox folder
  • Jan to send Priti types of Brec1 constructs
    • Are there maps of each of these constructs to share?
  • Priti to send protocols/antibody list for staining of CD7
  • Andrew Atkins (Ndhlovu Lab) – Set a subgroup meeting on CRISPR guide design 
    • Discuss the best method to design guides for CRISPR-BE
    • Subgroup: Zichong (Greene Lab), Bharath (Ott Lab), Hesong (Kumar Lab), Andrew (Ndhlovu lab)
    • Subgroup will present guide design method at the meeting on Dec 6th
  • Warner to send Priti a list of dCas9 vectors
  • Jan  to share the Tat-Tre Recombinase expression paper with Niren
  • Daniela (Ott Lab) – to generate a document on what JLats are available
  • Priti to start working on Brec1 delivery using a lenti or VLP based methods
  • Niren to start working on Brec1 using a mRNA delivery method

Please make sure to copy Danielle Lyons and Sydney Norman on emails so that we can assist in uploading documents to Dropbox. Emails to the full RF3 group can be made to HOPE-RF3-members@gladstone.ucsf.edu 

The next meeting will be on November 1st; Niren will present on mRNA delivery. The second presenter will be a member with new interesting data they would like to present. If you have any new data you would like to present at the next meeting please reach out to RF3 leaders, Melanie and Priti.

Going forward please keep your camera on during RF3 meetings when slides are not being shown in order to facilitate discussion.